1. Field of the Invention
The invention relates generally to the field of molecular biology. More specifically, the invention relates to improved methods for plant genetic transformation and compositions for achieving the same.
2. Description of Related Art
Transformation of plant cells by an Agrobacterium-mediated method involves exposing plant cells and tissues to a suspension of Agrobacterium cells that contain certain DNA plasmids. These plasmids have been specifically constructed to contain transgenes that will express in plant cells (see, for example, U.S. Pat. No. 5,034,322). Most often, one or more of the transgenes is a positive selectable marker transgene that permits plant cells to grow in the presence of a positive selection compound, such as an antibiotic or herbicide. These cells can be further manipulated to regenerate into whole fertile plants.
The methods for introducing transgenes into plants by an Agrobacterium-mediated transformation method generally involve a T-DNA (transfer DNA) that incorporates the genetic elements of at least one transgene and transfers those genetic elements into the genome of a plant. The transgene(s) are typically constructed in a DNA plasmid vector and are usually flanked by an Agrobacterium Ti plasmid right border DNA region (RB) and a left border DNA region (LB). During the process of Agrobacterium-mediated transformation, the DNA plasmid is nicked by an endonuclease, VirD2, at the right and left border regions. A single strand of DNA from between the nicks, called the T-strand, is transferred from the Agrobacterium cell to the plant cell. The sequence corresponding to the T-DNA region is inserted into the plant genome.
The integration of the T-DNA into the plant genome generally begins at the RB and continues to the end of the T-DNA, at the LB. However, the endonucleases sometimes do not nick equally at both borders. When this happens, the T-DNA that is inserted into the plant genome often contains some or all of the plasmid vector DNA. This phenomenon is referred to as “border read-through.” It is usually preferred that only the transgene(s) located between the right and left border regions (the T-DNA) is transferred into the plant genome without any of the adjacent plasmid vector DNA (the vector backbone). The vector backbone DNA contains various plasmid maintenance elements, including for example, origin of replications, bacterial selectable marker genes, and other DNA fragments that are not required to express the desired trait(s) in commercial crop products.
Considerable resources are directed at screening the genome of transgenic crop plants for the presence the vector backbone DNA. Methods such as polymerase chain reaction (PCR) and Southern blot analysis are most often employed to identify the extraneous vector backbone DNA. These methods are time consuming and expensive for large-scale screening work. The transgenic plants that are found to contain vector backbone DNA are generally not preferred for commercialization. Further, transgenic plants containing more than two transgenes are usually of little value for commercial development. Substantial efforts are expended regenerating plants from plant cell culture that have no commercial potential.
Thus, it would be of great benefit if methods and compositions could be developed that would greatly reduce the occurrence of vector backbone DNA in the genome of transgenic plants and/or increase the frequency of low copy transformation events. Fewer transgenic plants would have to be produced if a greater number were free from vector backbone DNA and most plants have one or two copies of the transgenes, greatly increasing the efficiency of transgenic plant production.